The endothelium is an important component of normal vascular homeostasis and endothelial dysfunction is a prelude to the development of vascular disease and its clinical manifestations such as heart attack and stroke. Endothelial dysfunction is thought to result, in part, from the injurious actions of established vascular disease risk factors such as hypercholesterolemia, hypertension, and diabetes. Resistance to endothelial dysfunction and injury is an important protective mechanism against vascular disease, as patients with preserved endothelial function are not predisposed to clinical vascular events. However, the precise molecular events that determine susceptibility or resistance to endothelial dysfunction are not known. Preliminary data presented in this application indicate that induction of a metabolic stress response (via AMP kinase activation) stimulates mitochondrial biogenesis and protects the endothelium from injurious stimuli. Furthermore, our data link this protective effect to suppression of c-Jun N-terminal kinase activation - a key component of the environmental stress response. Therefore, our central hypothesis is that the metabolic stress response and resultant stimulation of mitochondrial biogenesis are key determinants of endothelial cell phenotype and resistance to injurious stimuli. The objective of this application, therefore, is to determine the molecular mechanism(s) whereby metabolic stress and mitochondrial biogenesis modulate endothelial function and test the hypothesis that increased endothelial cell resistance to dysfunction will ameliorate the development of vascular disease. In order to achieve this objective, we will first determine the component(s) of the metabolic stress response and mitochondrial biogenesis required to protect the endothelium from injurious stimuli. We will model metabolic stress using pharmacologic (AICAR, 2-deoxyglucose) and molecular (PGC-11, RIP140) means and quantify mitochondrial biogenesis. We will then dissociate the metabolic stress response from mitochondrial biogenesis using pharmacologic (chloramphenicol) and molecular (Tfam) tools and determine the implications for endothelial protection. We will then probe the involvement of known metabolic targets such as eNOS, FOXOs, and SIRT1in endothelial stress resistance. Next we will determine the mechanism(s) whereby the metabolic stress response and mitochondrial biogenesis attenuates JNK activation. Metabolic stress and mitochondrial biogenesis will be manipulated and we will examine the implications for JNK activation by investigating important upstream (MAP3K and MAP2K) kinases as well as the specific JNK isoforms involved via a chemical genetic approach. We will then examine important determinants of JNK inactivation such as ROS and MAP kinase phosphatases. Finally, using a chemical genetic approach, we will determine temporal aspects of JNK regulation. Finally, we will determine the implications of manipulating endothelial cell mitochondrial biogenesis on endothelial dysfunction and vascular disease in vivo. We have developed tools to manipulate endothelial cell PGC11 as a model of mitochondrial biogenesis and mass in vivo. Using these animals, we will determine the implications of endothelial cell PGC11 on mitochondrial biogenesis and mass and endothelial resistance to the dysfunction associated with hypertension and atherosclerosis. The experiments outlined above should provide us with a solid working knowledge of how mitochondrial biogenesis and increased mitochondrial mass contributes to the control of endothelial phenotype and how this translates into homeostatic responses in vivo. With this information in hand, we should have the requisite insight to design new tools directed at modulating vascular redox status and phenotype with an eye toward the treatment of vascular disease.